Reovirus Neuropathogenesis

呼肠孤病毒神经发病机制

• 批准号: 10607594
• 负责人: TERENCE S. DERMODY
• 金额: $ 56.55万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-22 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607594
• 关键词: 3-Dimensional; Adult; Afferent Neurons; Atomic Force Microscopy; Axon; Axonal Transport; Binding; Biophysics; Brain; Brain imaging; CRISPR-mediated transcriptional activation; Capsid; Capsid Proteins; Cells; Cerebellum; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Cryoelectron Microscopy; Cultured Cells; Development; Disease; Double Stranded RNA Virus; Dynein ATPase; Electron Microscopy; Encephalitis; Engineering; Fiber; Genome; Hippocampus (Brain); Human; Immunocompetent; Immunoglobulins; In Vitro; Infection; Integration Host Factors; Integrins; Interneurons; Intervention; Intramuscular; Knockout Mice; Knowledge; Malignant Neoplasms; Mammals; Mediating; Morbidity - disease rate; Motor; Mus; Mutagenesis; Nervous system structure; Neuraxis; Neurons; Neuropathogenesis; Neurotropism; Newborn Infant; Oncolytic; Pattern; Peripheral; Pharmacology; Population; Process; Proteins; Reovirus; Reovirus Infections; Research; Resolution; Role; Route; Sensory Receptors; Series; Serotyping; Structure; Synapses; System; Testing; Thalamic structure; Tissues; Tropism; Viral; Viral Encephalitis; Viral Pathogenesis; Virulence; Virus; Work; blind; cell type; clinical development; experimental study; fast axonal transport; in vivo; mortality; mutant; nervous system disorder; neural circuit; neuronal cell body; neurotropic; neurotropic virus; neurovirulence; new therapeutic target; oncolytic vector; particle; receptor; receptor function; reconstruction; recruit; relating to nervous system; reverse genetics; screening; targeted treatment; transmission process; uptake; virus host interaction

Viral encephalitis is an important cause of morbidity and mortality in children and adults. Key knowledge gaps about the pathogenesis of viral encephalitis include how neurotropic viruses target the central nervous system (CNS), internalize into neurons, and disseminate in the brain. The proposed research uses reovirus, a genetically tractable double-stranded RNA virus that causes encephalitis and shows promise as an oncolytic agent, to dissect mechanisms of viral tropism, cell entry, and spread in the CNS. Reovirus strains display serotype-specific patterns of systemic dissemination and neurologic disease. Serotype 3 (T3) reoviruses are exquisitely neurotropic, internalize into neurons using macropinocytosis, and spread intracellularly by fast axonal transport. Reovirus uses β1 integrin to internalize into some types of cells, but its function in neural cell entry is unknown. Other previously identified reovirus receptors are dispensable for reovirus infection in the murine CNS. We recently identified a new reovirus receptor, paired immunoglobulin-like receptor B (PirB), that allows serotype-independent reovirus infection of cultured cells but is required for efficient T3 infection of neurons and full neurovirulence. Three integrated specific aims are proposed to enhance knowledge of reovirus neuropathogenesis that may have broader applications to other neurotropic viruses. In Specific Aim 1, functions of host receptors in reovirus neuropathogenesis will be determined. Biophysical interactions between PirB and reovirus will be assessed using cryo-electron microscopy, atomic-force microscopy, and structure- guided mutagenesis. The function of PirB in reovirus disease will be elucidated by comparing infection and virulence in wild-type (WT) and PirB-null mice. T3 reovirus-specific receptors that couple with PirB for neurotropism will be identified using CRISPR activation screening. In Specific Aim 2, mechanisms of reovirus entry into CNS neurons will be defined. The functions of PirB and β1 integrin in reovirus neural entry will be determined using mutant receptors and receptor-blind viruses. Receptor-dependent control of macropinocytosis will be elucidated using super-resolution, live-cell, and electron microscopy. Mechanisms of dynein motor recruitment for reovirus transport within axons will be dissected by investigating interactions between WT and mutant reovirus receptors and dynein subunits. In Specific Aim 3, the basis of reovirus dissemination in the CNS will be elucidated. The role of host receptors in reovirus neural transit will be defined using whole-brain imaging and three-dimensional reconstruction to trace reovirus infection and neural spread in WT and receptor- null mice. Mechanisms of reovirus transsynaptic transmission will be elucidated using in vitro and in vivo neural circuits. The effect of altering synaptic activity on reovirus neural transit will be tested using pharmacologic intervention and chemogenetically altered mice. Taken together, these studies will define mechanisms used by neurotropic reovirus to infect and disseminate in the brain and may promote further development of reovirus oncolytics that selectively target the nervous system.

病毒性脑炎是导致儿童和成人发病和死亡的重要原因。关键知识差距 关于病毒性脑炎的发病机制，包括嗜神经病毒如何攻击中枢神经系统 中枢神经(CNS)，内化到神经元，并在大脑中传播。拟议的研究使用了呼肠孤病毒，一种 基因上易受感染的双链RNA病毒，可引起脑炎并显示出作为溶瘤药物的希望 分析病毒的嗜性、细胞进入和在中枢神经系统中传播的机制。呼肠孤病毒株显示 全身性传播和神经系统疾病的特定血清型模式。血清3型(T3)呼肠孤病毒是 微妙的嗜神经性，通过巨噬细胞吞噬作用内化为神经元，并通过FAST在细胞内传播 轴突运输。呼肠孤病毒使用β1整合素内化到某些类型的细胞中，但其在神经细胞中的功能 条目未知。以前发现的其他呼肠孤病毒受体对于呼肠孤病毒感染在 小鼠中枢神经系统。我们最近发现了一种新的呼肠孤病毒受体，配对免疫球蛋白样受体B(PirB)， 允许培养细胞感染非血清型呼肠孤病毒，但对有效的T3感染是必需的 神经元和完全的神经毒性。为加强对呼肠孤病毒的认识，提出了三个综合的具体目标。 神经致病机制可能对其他嗜神经病毒有更广泛的应用。在具体目标1中， 宿主受体在呼肠孤病毒神经发病机制中的作用将被确定。之间的生物物理相互作用 PirB和呼肠孤病毒将使用冷冻电子显微镜、原子力显微镜和结构- 引导诱变。PirB在呼肠孤病毒病中的作用将通过比较感染和 野生型(WT)和PirB缺失小鼠的毒力。与PirB结合的T3呼肠孤病毒特异性受体 趋神经性将通过CRISPR激活筛查来确定。在特定目标2中，呼肠孤病毒的机制 进入中枢神经系统神经元将被定义。PirB和β1整合素在呼肠孤病毒神经进入中的作用 使用突变受体和受体失明病毒来确定。巨噬细胞增多症的受体依赖性调控 将使用超分辨率、活细胞和电子显微镜来阐明。动力蛋白马达的作用机制 通过研究WT和WT之间的相互作用，将剖析轮状病毒在轴突内运输的招募 突变的呼肠孤病毒受体和动力蛋白亚基。在具体目标3中，呼肠孤病毒在中国传播的基础 中枢神经系统将被阐明。宿主受体在呼肠孤病毒神经传递中的作用将通过全脑来确定。 图像和三维重建追踪呼肠孤病毒感染和神经扩散在WT和受体- 零老鼠。利用体外和体内神经细胞阐明呼肠孤病毒跨突触传递的机制 电路。改变突触活性对呼肠孤病毒神经传递的影响将通过药理学进行测试 干预和化学遗传学改变的小鼠。综上所述，这些研究将定义 嗜神经性呼肠孤病毒在脑内感染和传播，并可能促进呼肠孤病毒的进一步发展 选择性地以神经系统为靶点的溶瘤药物。